Plasma Coagulation Inhibitors
DRAFT Subcommittee Minutes
23 July 2011
14:00-18:00
Room B-2
Chairman: Steve Kitchen (UK)
Co-chairmen: Elisabetta Castoldi (NL), Elaine Gray (UK), Tilman Hackeng (NL), Richard Marlar (US), Piet Meijer (NL), Laurent Mosnier (US)
Educational programme _ Chairs S Kitchen, E Gray.
Thrombophilia: Who should be tested?
Trevor Baglin (UK)
APC resistance: biological basis and acquired influences
Elisabetta Castoldi , Maastricht, The Netherlands
Problems with Laboratory Assays for Protein C, Protein S and Antithrombin.
Richard A. Marlar, USA
Minutes/Summaries
Thrombophilia: Who should be tested?
Trevor Baglin (UK)
The evidence related to risk of recurrence after provoked and unprovoked VTE was reviewed and the rate that would justify anticoagulation was discussed. It was concluded that investigation of a patient for heritable thrombophilia should be the exception rather than the rule. Recent UK guidelines based on the available published evidence from studies in many countries had recommended that indiscriminate testing for heritable thrombophilia in unselected patients presenting with a first episode of venous thrombosis is not indicated (strong evidence) and that decisions regarding duration of anticoagulation in unselected patients should be made with reference to whether or not a first episode was unprovoked (strong evidence). The same document also recommends that testing for heritable thombophilia in unselected patients with a strong family history or unprovoked recurrent thrombosis may influence decisions though a validated recommendation on patient selection is not currently possible. Case finding of asymptomatic relatives with low risk of thombophilia such as FVLeiden or F2G20210A is not indicated.
APC resistance: biological basis and acquired influences
Elisabetta Castoldi , Maastricht, The Netherlands
The protein C pathway is a pivotal anticoagulant system responsible for the proteolytic inactivation of coagulation factors (F) Va and VIIIa, the essential cofactors of the prothrombinase and intrinsic tenase complexes, respectively. Cleavage of FVa and FVIIIa by activated protein C (APC) is stimulated by the APC-cofactors protein S and FV.
APC resistance is defined as a poor response of plasma to the addition of exogenous APC in vitro (Dahlbäck et al, PNAS 1993). Although it was initially discovered in a single thrombotic patient, it was soon recognised as the most prevalent risk factor for venous thrombosis. Most cases of APC resistance are attributable to the FV Arg506Gln mutation (FV Leiden) (Bertina et al, Nature 1994), which abolishes one the APC-cleavage sites in FV(a). However, several other genetic and acquired factors contributing to APC resistance have been described in recent years, most of which act by altering the levels of coagulation factors and inhibitors modulating APC activity. The numerous APC resistance assays currently available are based on different principles and are therefore differentially sensitive to the manifold determinants of APC resistance.
This educational lecture reviewed the multi-factorial etiology of APC resistance and discussed its implications for thrombosis risk and for APC resistance testing with different functional assays.
Problems with Laboratory Assays for Protein C, Protein S and Antithrombin. Richard A. Marlar, USA
PC, PS and AT assays are the most common assays performed for the evaluation of patients with potential thrombophilia. These assays appear to be essentially straight forward in their methodology. However there are numerous direct and indirect parameters that can affect these assays and their reported results. External variation of the plasma samples (pre-analytical variables), assay methodology differences (analytical variables), and differences in reference intervals (post-analytical variables) can complicate the diagnostic capabilities of the laboratory. This is on top of evaluation that do not take into account the potential complex genetic abnormalities that the assays are trying to accurately detect. This presentation will review the sample and assay variables that can be troublesome for proper PC, PS and AT assay performance and a brief discussion of genetic defects that can hamper assay performance. Finally, recommendations for the best assay methods will be presented along with the best protocol for confirming the positive diagnosis of one of these thrombophilic deficiencies.
Subcommittee Business section
Session 1 Chairs P Meijer, R Marlar
Instability of Protein S in blood samples.
Kieron Hickey UK
Whole blood samples from normal donors were stored at room temperature for up to 72 hours prior to processing (centrifugation at 2000G for 10 minutes). Plasma aliquots were frozen at -80 oC and assays of free protein S (FPS) performed. The FPS antigen as determined by latex immunoassay utilises beads coated with C4B binding protein to capture FPS and a monoclonal antibody to human protein S to initiate aggregation. In 10 normal donors there was a 14% reduction in FPS at 24 hours (relative to time 0), rising to 37% at 72 hours. In contrast FPS antigen determined by ELISA (method of Woodhams) was unchanged in that time.
A second study utilised PS activity assays from Siemens, Instrumentation Laboratory and Precision Biologic. Whole blood samples were stored for up to 24 hours at room temperature prior to processing. For all three methods there was a reduction of 3 to 5% in PS activity after 4 hours, rising to a 13- 15% mean loss after 24 hours.
In this second study FPS antigen by latex immumoassay was reduced by 6% at 12 hours and 11% at 24, confirming the findings of the first study. ELISA Free PS antigen again demonstrated no change.
Elaine Gray, NIBSC, UK
Data on the calibration of SSC plasma standard lot 4 were presented. All analytes were calibrated against relevant WHO International Standards. SSC plasma standard lot 3 was included in the exercise for an assessment of continuity and stability of lot 3. Overall there was good agreement between unitage for lot 3 between the original calibration and the current study although the free PS Ag was associated with a difference of 8% thought to relate to the replacement of the WHO international standard (from first to second).
In relation to lot 4 for PC activity there was no significant difference between clotting and chromogenic PC assay data. Inter-laboratory variability was assessed by geometric coefficient of variation (GCV) which was good for PC antigen (6.9%) and activity (3.6%). The PS activity data included 5 sets of Instrumentation Laboratory Pro S results and 6 sets of Staclot data. The difference between results with these two methods was significant but the overall inter-laboratory GCV was only 7% and participants had approved the proposed assigned single value including all results. Free PS data included 10 sets of latex immunoassay results and 7 sets of ELISA result. There was no significant difference with a GCV of 6.8%. Total PS assay data was also precise (GCV 3.8%). For AT there was no significant difference between results with IIa and Xa based assays and a single value was assigned. AT antigen assays were acceptably precise. All participants had agreed with all the proposed potencies. There were no objections to these assigned potencies from the Plasma coagulation inhibitors subcommittee.
Protein S assays: need for standardisation
Ian Jennings, UK NEQAS (Blood Coagulation) UK
UK NEQAS (Blood Coagulation) data show a fall in centres performing PS activity assays between 2001-2010, with a concomitant rise in free PS assays. The latter is entirely due to laboratories performing latex-based assays. Between-laboratory agreement for free PS antigen assays is good, with CVs around 10% in proficiency testing exercises. However, for PS activity assays, marked differences between results obtained with different kits are still seen. These differences are apparent for samples distributed in both NEQAS and ECAT surveys, and could not be explained in an in-house investigation. A multicentre study investigating PS activity in both frozen and lyophilised plasmas is planned. This will be submitted for approval as a subcommittee project.
Biological Variation for AT, PC and PS: their relation to analytical quality
Piet Meijer (ECAT, The Netherlands)
Sources of analytical variation were discussed including pre analytical factors related to the subject or sample collection and handling. Analytical variability could be random involving inherently unpredictable variations in test results or systematic errors which are typically constant or proportional to the test result. Data from a study of biological variation were presented. This involved 15 samples collected over 1 year from each of 40 normal subjects. The within subject CV was 4 to 9% for AT,PC and PS determinations. Analytical variation of these assays was in the range 1 to 6%. Data on long term analytical imprecision using results of the ECAT quality assurance program indicated CVs of around 7% for chromogenic assays of AT and PC, with CVs of 10 to 16% for clot based and antigen assays of PC and PS.
Evaluation of a novel total Protein S assay system for screening of protein S type II deficiency
Prof Hiroko Tsuda Japan
A novel assay system for activity and antigen levels of total protein S (PS) for automated analyzers was described. To test usability of this assay to screen for type II PS deficiency in Japanese venous thromboembolism (VTE) plasma samples of healthy volunteers and confirmed type II deficiency patients was assessed. Reference intervals (average ± 2SD) of healthy male (n = 98) were 19.3 - 32.8 μg/mL for total PS antigen and 18.8 - 32.3 μg/mL [PS equivalent] for total PS activity, respectively, and those of female (n = 89) being 16.0 - 29.0 μg/mL and 14.8 - 28.8 μg/mL [PS equivalent], respectively. The mean specific activity for healthy individuals (n = 187) was 0.99 and limit of reference interval was 0.79. Specific activities were found to be < 0.69 for specimens from patients with confirmed PS type II deficiency and those administered warfarin. The automated total PS assay system was considered an effective screening tool for PS type II deficiency,
Session 2 Chairs Elisabetta Castoldi , Tilman Hackeng
Mutation databases for PC, PS and AT.
Carolina Pintao (The Netherlands)
Hereditary deficiency of the coagulation inhibitors protein C, protein S and antithrombin is rare and associated with increased risk of thrombosis. The genetic basis of the deficiencies is highly heterogeneous, almost every family segregating a different mutation.
The exponential growth of data generated by high throughput sequencing and genome-wide association studies, as well as the growth of knowledge and understanding of the relationship between genotype and phenotype increases the need for constantly updated and comprehensive mutation databases.
Two mutation databases currently aim to provide information on genetics with different overlapping priorities: the Online Mendelian Inheritance in Man (OMIM), and the Human Gene Mutation Database (HGMD) (Cardiff, UK). The first is aimed on genotype-phenotype knowledge, but is not comprehensive on adding variations, focusing on the first and most relevant ones. The second is aimed on adding every public available mutation associated with a gene, but has little phenotypic information (Samuels and Rouleau, Nat Rev Genet, 2011).
In contrast, the so-called locus-specific databases (LSDs), which are typically curated by experts on a particular area, aim to provide comprehensive information associated with a particular gene or disease. Databases following this model have been released before for protein C (1995), antithrombin (1997), and protein S (2000) deficiency, on behalf of the International Society of Thrombosis and Haemostasis.
This presentation is aimed to discuss the need to update (and maintain) the databases of mutations for protein C, antithrombin and protein S deficiencies.
